Infectious diseases in developing countries continue to be one of the main causes of death, exceeding deaths from cardiovascular diseases and cancer. Infectious diseases include those provoked by facultative intracellular microorganisms, as is the case of tuberculosis (TB), which is the main cause of death among these diseases. The appearance of multi-resistant strains and the Acquired Immune Deficiency Syndrome (AIDS) have increased the incidence of this disease (Kochi A. The global tuberculosis situation and the new control strategy of the World Health Organization. Tubercle 1991, 72:1-6).
TB can also affect animals, principally domestic and farm animals such as cows, cats, dogs and fowl. Although other diseases, such as trypanosomiasis and leishmaniasis, represent a public health problem, their incidence is not as important as that of TB, but in all these diseases treatment is complicated due to the immunological changes patients present.
The AIDS pandemic has caused cases of pulmonary and extra-pulmonary tuberculosis to increase over the last decade, placing it as a priority disease for health programs in almost the whole world (Kochi A. The global tuberculosis situation and the new control strategy of the World Health Organization. Tubercle 1991, 72:1-6. Sudre P, ten Dam G, and Kochi A. Tuberculosis: a global overview of the situation today. Bull World Health Organ 1992; 70:149).
A person infected with the human immunodeficiency virus (HIV) has 10 times the risk of developing tuberculosis. In an HIV infected individual, the presence of other infections, including tuberculosis, can permit the virus to multiply more rapidly resulting in an accelerated progression of the infection. As the infection progresses, leukocytes decrease in number and function, the immune system is no longer able to prevent the growth and dissemination of Mycobacterium tuberculosis. 
It is calculated that one third of the world population is infected with Mycobacterium tuberculosis. After infection, the risk of developing the disease is approximately 10%, and the remaining 90% continue with latent infection from the viable bacillus. This 10% amounts to 8 million persons reported each year with active tuberculosis, resulting in 3 million deaths a year (Kochi A. The global tuberculosis situation and the new control strategy of the World Health Organization. Tubercle 1991, 72:1-6. Sudre P, ten Dam G, and Kochi A. Tuberculosis: a global overview of the situation today. Bull World Health Organ 1992; 70:149). This disease is, moreover, a serious problem faced by patients undergoing hemodialysis.
Tuberculosis treatment is long term, and hence dropping-out is frequent. This has led to a reactivation of the disease and the appearance of strains that are multi-resistant to the medicines normally used. (isoniazid, ethambutol, pyrazinamide, rifampicin and a derivative of the latter, rifapentin)
Transforming Growth Factor-beta Type (TGF-β)
TGF-β is the prototype of a superfamily of autocrine and paracrine factors that participate in the control of embryonic development, cell differentiation and proliferation, tissue repair and regulation of the immune system. TGF-β is a potent inhibitor of cell proliferation in lymphoid and epithelial lineages and failure in this antiproliferative response has been associated with the formation of malignant tumors, supporting the idea that TGF-β and its signal transducing molecules are authentic “tumor suppressors. TGF-β plays an important role in tissue repair.
Platelets release TGF-β in the site of a lesion, setting off a cascade of events leading to repair of the wound, a process in which TGF-β plays a preponderant role (Massagué, J. (1990). The transforming growth factor-β family. Annu Rev Cell Biol 6, 597-641. Massagué, J., Blain, S. W., and Lo, R. S. (2000). TGF-β signaling in growth control, cancer, and heritable disorders. Cell 103, 295-309)
Among the interleukins, TGF-β is a unique cytokine because it efficiently suppresses cell immunity by acting at several levels (Letterio J J, Roberts A B. Regulation of immune response by TGF-β. Annu Rev Immunol 1998; 16:137-61). As happens with many other types of cells, TGF-β inhibits the proliferation of lymphocytes, above all mature T cells that have already been activated (Kehrl J H, Wakefield L M, Roberts A B, Jakowlew S, Alvarez-Mon M, Derynck R, Sporn M, Fauci A S. Production of transforming growth factor beta by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med 1986; 163: 1037-50), while virgin or inactivated T lymphocytes are relatively resistant to the antimitogenic effect of TGF-β. TGF-β also inhibits the proliferation of B lymphocytes and induces their cell death by apoptosis (Lomo J, Blomhof H K, Beiske K, Stokke T, Smeland E B. TGF-β1 and cyclic AMP promotes apoptosis in resting human B lymphocytes. J Immunol 1995; 154:1634-43) and suppresses the cytolytic differentiation and activity of NK and T cells (Letterio J J, Roberts A B. Regulation of immune response by TGF-β. Annu Rev Immunol 1998; 16:137-61). Another important inhibitory effect of TGF-β is suppression of the expression of the major histocompatibility complex (MHC) class 2 molecules in macrophages (Czarniecki C W, Chiu H H, Wong G H, Mc Cabe S M, Palladino M A. Transforming growth factor beta 1 modulates the expression of class II histocompatibility antigens on human cells. J Immunol 1988; 140:4217-4232), thus interfering in the antigen presentation process, avoiding T lymphocyte activation. The most important result of this interference in T cell activation is the inhibition of the secretion of interleukin 2 (IL-2), because this cytokine is an essentially inducing factor of cell proliferation. Indeed, it is considered that this is the principal mechanism through which TGF-β inhibits lymphocyte proliferation. Another T lymphocyte mitogenic cytokine and macrophage activator is interleukin 1 (IL-1).
TGF-β also directly and indirectly inhibits its production by suppressing its receptor specific expression and, at the same time, increasing the release of the IL-1 soluble receptor antagonist whose function is to trap and avoid the binding of this cytokine to its receptor (Turner M, Chantry D, Katsikis T, Berger A, Brennan F M, Feldman M. Induction of interleukin 1 receptor antagonist protein by transforming growth factor beta. J Immunol 1991; 21:1635-1639)
One of the fundamental suppressor effects of TGF-β on the immune system is the deactivation of macrophages, which can be carried out through direct inhibition of the production of oxygen free radicals and nitric oxide or, indirectly, by suppressing the production of macrophage activating cytokines such as the tumor necrosis factor alpha (TNF-α) and interferon gamma (INF-γ) and their receptors (Ranges G E, Figari I S, Espevik T, Palladino M A. Inhibition of cytotoxic T cell development by transforming growth factor beta and reversal by recombinant tumor necrosis factor alpha. J Exp Med 1987; 166:991-999. Pinson D M, Le Claire R D, Lorsbach R B, Parmely M J, Russell R. Regulation by transforming growth factor beta-1 of expression and function of the receptor for INF gamma on mouse macrophages. J Immunol 1992; 149:2028-2038). For the production of nitric oxide, it is necessary for TNF-α and INF-γ to activate the inducible nitric oxide synthetase enzyme (iNOS), and TGF-β inhibits both the transcription and translation of the gene encoding this enzyme (Vodovotz Y, Bogdan C. Control of nitric oxide synthase expression by transforming growth factor beta: implications for homeostasis. Prog Growth Factor Res 1994; 5:341-351). Furthermore, the participation of INF-γ is fundamental for the activation of macrophages as part of the Th-1 response, and one of the most efficient immunosuppressor effects of TGF-β is to inhibit the production of INF-γ and its receptor expressed on the macrophage membrane (Pinson D M, Le Claire R D, Lorsbach R B, Parmely M J, Russell R. Regulation by transforming growth factor beta-1 of expression and function of the receptor for INF gamma on mouse macrophages. J Immunol 1992; 149:2028-2038). TGF-β is an efficient promoter of Th-2 cytokines, particularly interleukin 10 (Schmitt E, Hoehn P, Huels C, Goedert S, Palm N, Rude E, Germann T. T helper type 1 development of naive CD-4 T cells requires the coordinated action of interleukin 12 and interferon gamma and is inhibited by transforming growth factor beta. Eur J Immunol 1994; 24:793-798. Strober W, Kelsall B, Fuss I, Marth T, Ludviksson B, Ehrhardt R, Neurath M. Reciprocal IFN gamma and TGF-β responses regulate the occurrence of mucosal inflammation. Immunol Today 1997; 18:61-64. Maeda Y, Kuwahara H, Ichimura Y, Ohtsuki M, Kurakata S, Shirahishi A. TGF-β enhances macrophage ability to produce IL-10 in normal and tumor bearing mice. J Immunol 1995; 15:49264932).
Sustained, excessive production of TGF-β has been implicated as an important pathogenic factor in the fibrosis and tissue damage present in different diseases (Wahl S M. Transforming growth factor beta: the good, the bad and the ugly. J Exp Med 1994; 180:1587-90). But apart from this important fibrosis inducing effect, in several chronic inflammatory diseases, such as rheumatoid arthritis, leprosy and tuberculosis, there is an anergy of cell immunity which as been attributed in part to excessive production of TGF-β (Letterio J J, Roberts A B. Regulation of immune response by TGF-β. Annu Rev Immunol 1998; 16:137-61. Wahl S M. Transforming growth factor beta: the good, the bad and the ugly. J Exp Med 1994; 180:1587-90. Tossi Z, Ellner J. The role of TGF-β in the pathogenesis of human tuberculosis. Clin Immunol Immunopathol 1998; 87:107-114). This pathogenic proposition has been strengthened by observations made in transgenic animals with overexpression of the TGF-β gene that suffer intense immunosuppression and also by the extensive multifocal inflammation presented by mice with disruption of the gene coding for TGF-β1 (Shull M M, Ormsby I, Kier A B, Pawloski S, Diebold R, Yin M, Allen R, Sidman C, Proetzel G, Calvin D. Targeted disruption of the mouse transforming growth factor beta 1 gene results in multifocal inflammatory disease. Nature 1992; 359:693-99). In animal models of infections from facultative intracellular germs such as leishmaniasis and trypanosomiasis, it has been observed that TGF-β is produced in excess favoring progression of the disease (Barral Netto M, Barral A, Brownell C E, Skeiki Y A, Ellingsworth L R, Twardzic D R, Reed S G. Transforming growth factor beta in leshmanial infection; a parasite escape mechanism. Science 1992; 257:545-48. Gazzinelli R T, Oswald I P, Hieny S, James S L, Sher A. The microbicidal activity of interferon gamma treated macrophages against Trypanosoma cruzi, involves an L-arginine-dependent, nitrogen oxide-mediated mechanism inhibitable by interleukin 10 and transforming growth factor beta. Eur J Immunol 1992; 22:2501-06). In human leishmaniasis and filariasis, the participation of TGF-β has also been documented in immunosuppression induction (Barral Neto M, Barral A, Brodskyn C Carvalho E M. Cytotoxicity in human mucosal and cutaneous leshmaniasis. Parasite Immunol 1995, 17:21-28. King C L, Mahanty S, Kumarasawami V, Abrams J S, Regunthan J, Jeyamaran K, Ottesen E A, Nutman T B. Cytokine control of parasite specific anergy in human lymphatic filariasis. J Clin Invest 1993, 92:1667-1673). In relation to viral infections, it has been observed that progressive immunodeficiency during AIDS is associated with a gradual increase of TGF-β (Lotz M, Seth P. TGF beta and HIV. Annu NY Acad Sci 1993, 685:501-511), and in cases of HIV and M. tuberculosis co-infection there is a synergistic potentiating effect on the production of TGF-β which increases viral activity accelerating the disease (Maltman J, Pargnell I B, Graham G J. Specificity and reciprocity in the interactions between TGF-β, and macrophage inflammatory protein 1-alpha. J Immunol 1996, 156:1566-1571).
Mycobacterium tuberculosis, its purified protein derivatives (PPD) and some of their molecular components, such as lipoarabinomannan, induce the macrophages to produce and secrete large amounts of TGF-β (Tossi Z, Ellner J. The role of TGF-β in the pathogenesis of human tuberculosis. Clin Immunol Immunopathol 1998; 87:107-114). The participation of INF-γ is fundamental in the activation of macrophages as part of the Th-1 response which is essential in the control of infectious diseases produced by facultative intracellular germs such as tuberculosis and leshmaniasis (Hernández Pando R, Orozco E H, Arriaga K, Sampieri A, Larriva Sahd J, Madrid M V. Analysis of the local kinetics and localization of interlukin 1 alpha, tumor necrosis factor alpha and transforming growth factor beta during the course of experimental pulmonary tuberculosis. Immunology 1997; 90:607-17). Due to the fact that TGF-β is an efficient blocker of INF-γ production and, at the same time, induces cytokine Th-2 production (Schmitt E, Hoehn P, Huels C, Goedert S, Palm N, Rude E, Germann T. T helper type 1 development of naive CD-4 T cells requires the coordinated action of interleukin 12 and interferon gamma and is inhibited by transforming growth factor beta. Eur J Immunol 1994; 24:793-798. Strober W, Kelsall B, Fuss I, Marth T, Ludviksson B, Ehrhardt R, Neurath M. Reciprocal IFN gamma and TGF-β responses regulate the occurrence of mucosal inflammation. Immunol Today 1997; 18:61-64. Maeda Y, Kuwahara H, Ichimura Y, Ohtsuki M, Kurakata S, Shirahishi A. TGF-β enhances macrophage ability to produce IL-10 in normal and tumor bearing mice. J Immunol 1995; 15:4926-4932), it contributes to the progression of these diseases. In experimental models of pulmonary tuberculosis and in the active human disease, a high production of TGF-β has been shown (Hernández Pando R, Orozco E H, Arriaga K, Sampieri A, Larriva Sahd J, Madrid M V. Analysis of the local kinetics and localization of interlukin 1 alpha, tumor necrosis factor alpha and transforming growth factor beta during the course of experimental pulmonary tuberculosis. Immunology 1997; 90:607-17. Hirsch C, Ellner J, Blinkhorn R, Tossi Z. In vitro restoration of T cell responses in tuberculosis and augmentation of monocyte effector function against Mycobacterium tuberculosis by natural inhibitors of transforming growth factor beta. Proc Natl Acad Scie 1997; 94:3926-3931), which coincides with the decrease in protective immunological activity mediated by the cytokines produced by lymphocytes Th-1 (INF-γ, IL-2) and TNF-α. In vitro studies have shown that the anergy of cell immunity that generally exists in patients with active pulmonary tuberculosis can be corrected with the administration of TGF-β blocker antibodies or natural blockers, which significantly reduces intracellular growth of the tuberculosis bacillus (Hirsch C, Ellner J, Blinkhorn R, Tossi Z. In vitro restoration of T cell responses in tuberculosis and augmentation of monocyte effector function against Mycobacterium tuberculosis by natural inhibitors of transforming growth factor beta. Proc Natl Acad Scie 1997; 94:3926-3931). Indeed, TGF-β neutralizing antibodies or decorin as a TGF-β inhibitor have been used in in vitro studies to promote the efficiency of the immune system in eliminating M. tuberculosis. (Hirsch C, Ellner J, Blinkhorn R, Tossi Z. In vitro restoration of T cell responses in tuberculosis and augmentation of monocyte effector function against Mycobacterium tuberculosis by natural inhibitors of transforming growth factor beta. Proc Natl Acad Scie 1997; 94:3926-3931) However, these products have the disadvantage of evoking the formation of antibodies that block their activity or decrease the efficiency of the anti-inflammatory activity of TGF-β, discouraging the use of these inhibitors.
The TGF-β signaling pathway begins on the cell surface with the association, mediated by this ligand, of type I and II receptors, which can be considered heteromeric sub-units of the “signaling receptor”. Type I and II receptors are transmembrane proteins whose intracellular portions consist of serine and threonine protein kinases. The phosphorylation of the kinase of receptor I by the kinase of receptor II causes its activation and hence the phosphorylation of members of a novel family of proteins called “Smads”, which form heteromeric complexes that migrate to the nucleus to regulate the transcriptional events involved in TGF-β responses (Massagué, J. (1998). TGF-β signal transduction. Annu Rev Biochem 67, 753-791. Massagué, J., and Chen, Y.-G. (2000). Controlling TGF-β signaling. Genes and Development 14, 627-644). As well as the signaling receptor, other cell surface proteins have been identified that bind TGF-β. Betaglycan and endoglin are transmembranal proteins with large extracellular domains capable of binding to TGF-β and small intracellular regions, very similar the one to the other. Betaglycan is present in most tissues and cell lines, except in the endothelium, in contrast, endoglin is principally expressed in the latter. Although none of these glycoproteins seem to have a clear function in the TGF-β intracellular transduction pathway, both seem to modulate the extracellular access of the ligand to type I and II receptors (Massagué, J., and Chen, Y.-G. (2000). Controlling TGF-β signalling. Genes and Development 14, 627-644).
Betaglycan, also known as TGFβ, type III receptor is a transmembranal proteoglycan containing glycosaminoglycans (GAG) of the heparan and chondroilin sulfate type that belongs to a new class of receptors called “co-receptors”, because of their capacity to modulate ligand interaction with the signaling receptors. Even devoid of GAG, betaglycan binds the three different TGF-β isoforms with high affinity. Due to this high affinity, the presence of betaglycan compensates for the low affinity these signaling receptors have for the ligand, thus permitting equipotentiation of the different TGF-β isoforms. This effect seems to be mediated by the capacity of betaglycan to associate with TGFβ and receptor II in a tripartite complex of “ligand presentation” (Esparza-López, J., Montiel, J. L., Vilchis-Landeros, M. M., Okadome, T., Miyazono, K., and López-Casillas, F. (2001). Ligand binding and functional properties of betaglycan, a co-receptor of transforming growth factor-β superfamily. Specialized binding regions for transforming growth factor-β and inhibin A. J Biol Chem 276, 14588-14596). Although the capacity to equipotentiate the TGF-β isoforms is one of the best characterized functions of betaglycan, Barnett et al. demonstrated that the presence of betaglycan is indispensable for the adequate formation of the embryonic primordia that give origin to the cardiac valves (a process that depends on the presence of TGF-β), which leads to the belief that some of the regulating effects of TGF-β on the embryonic development depend on the direct participation of this co-receptor in signaling mechanisms (Brown, C. B., Boyer, A. S., Runyan, R. B., and Barnett, J. V. (1999). Requirement of type III TGF-β receptor for endocardial cell transformation in the heart. Science 283, 2080-2082). As well as the membranal form of betaglycan, there is a “soluble” form which is found in serum and extracellular matrixes. This has its origin in a juxtamembrane proteolytic escision that releases the receptor ectodomain from its anchorage to the membrane. The recombinant form of soluble betaglycan has been shown to have a opposite function to the membranal form, that is a potent neutralizing agent of the effects of TGF-β (López-Casillas, F., Payne, H. M., Andres, J. L., and Massagué, J. (1994). Betaglycan can act as dual modulator of TGF-β access to signaling receptors: mapping of ligand binding and GAG attachment sites. J Cell Biol 124, 557-568. Vilchis-Landeros, M. M., Montiel, J. L., Mendoza, V., Mendoza-Hernández, G., and López-Casillas, F. (2001). Recombinant soluble betaglycan is a potent and isoform-selective transforming growth factor-β neutralizing agent. Biochemical Journal 355, 215-222). These studies conducted in vitro with tissue cells in culture have made it possible to define betaglycan as an “extracellular switch” that modulates the effects of TGF-β (Attisano, L., Wrana, J. L., López-Casillas, F., and Massagué, J. (1994). TGF-β receptors and actions. Biochim Biophys Acta 1222, 71-80).
Prostaglandins
Prostaglandins are potent mediators of intercellular communication, and prostaglandin E2 (PGE2) in high concentrations acts as an immunosuppressor mechanism for immunity mediated by T cells (Phipps R P, Stein S H, Roper R L. A new view of prostaglandin E regulation of the immune response. Immunol Today 1991; 12:349-52). Certain cell signals are accompanied by a rapid rearrangement of the cell membrane lipids through the activation of lipases that generate bioactive lipids that can serve as intra and/or extracellular mediators. The most important of these lipids is arachidonic acid, a poly-unsaturated fatty acid of 20 carbons, that is normally esterified in phospholipids of the cell membrane and is released through the activation of cell phospholipases (Serhan C N, Haeggström J Z, Leslie C C. Lipid mediator networks in cell signaling: update and impact of cytokines. FASEB J 1996; 10:1147-58). The products derived from the metabolism of arachidonic acid are the so-called eicosanoides, which are considered to be autacoids because they are short scope local hormones that are rapidly formed and have a local effect and their activity falling spontaneously or by the effect of enzyme degradation. Eicosanoids affect many physiological and pathological events, they are synthesized by two major classes of enzymes: cyclooxygenases (COX) and lipoxygenases which produce, prostaglandins and leukotrienes, respectively (Phipps R P, Stein S H, Roper R L. A new view of prostaglandin E regulation of the immune response. Immunol Today 1991; 12: 349-52. Serhan C N, Haeggström J Z, Leslie C C. Lipid mediator networks in cell signaling: update and impact of cytokines. FASEB J 1996; 10:1147-58). The cyclooxygenase pathway is mediated by two different enzymes: COX-1, which is a constitutively expressed enzyme, and COX-2, which is a highly inducible enzyme expressed in the inflamed tissue after exposure to growth factors, cytokines and other inflammation mediators. Of the prostaglandins, the most extensively studied are those of series E (PGE). There is evidence showing that PGE-2 has an important immunosuppressor effect, including a decrease in lymphocyte proliferation, NK activation and MHC-II expression (Goto T R, Herberman R B, Maluish A, Strong D M. Cyclic AMP as a mediator of prostaglandin E induced suppression of human natural killer cell activity. J Immunol 1983; 130:1350-55. Snyder D S, Beller D I, Unanue E R. Prostaglandins modulate macrophage expression. Nature 1982; 299:163-65). It has also been found that PGE-2 play a predominant role in the regulation of the Th1 and Th2 type response. Indeed, PGE-2 inhibits the production of Th1 cytokines, interferon gamma, interleukin 2 and 12, blocks the activation of macrophages and suppresses the production of interleukin 1 and TNF-α (Betz M, Fox B S. Prostaglandin E2 inhibits production of Th-1 lymphokines but not of Th-2 lymphokines. J Immunol 1991; 146:108-13. Kuroda E, Suguira T, Zeki K, Yoshida Y, Yamashita U. Sensitivity difference to the suppressive effect of prostaglandin E2 among mouse strains: a possible mechanism to polarize Th2 type response in BALB/c mice. J Immunol 2000; 164: 2386-95). This activity of PGE-2 may be important in intracellular infections, since it has been shown in mice and humans that infection from mycobacteria is controlled by the activation of macrophages through the production of Th1 cytokines (Rook G A W, Hernández Pando R. Pathogenesis of tuberculosis. Annual Rev Microbiol 1996, 50:259-284).
Tuberculosis is a disease that is treated with antibiotics that need to be administered for 6 months in combination with 3 different antibiotics and this causes a high treatment drop-out frequency by patients generating frequent relapses and resistance to the antibiotics (Rook G A W, Hernández Pando R. Pathogenesis of tuberculosis. Annual Rev Microbiol 1996, 50:259-284). Thus, one alternative would be to design new treatment schemes making it possible to shorten the antibiotic therapy. Another excellent example of this situation occurs with leprosy.
One potentially useful therapeutic strategy in diseases with this immunological pattern is immunotherapy, that consists in administering substances that favor the protective immune response. Considering the prominent immuno-regulator role played by TGF-β and prostaglandins during infection by facultative intracellular microorganisms, we have developed the present invention with the purpose of assisting the treatment by developing the response of the organism to defend itself against facultative intracellular microorganisms, especially against the genus Mycobacterium, Leishmania, Trypanosoma among others, in an attempt to inhibit the immunosuppressor effect of TGF-β but, in turn, mitigating in another way, the pro-inflammatory side effect resulting from its inhibition.
For this reason, we have developed the present invention with the purpose of assisting the treatment of diseases caused by facultative intracellular organisms in order to reduce antibiotic treatment time and, in a secondary way, by shortening the application time for these treatments, decrease the drop-out percentage.